Engineered Nanostructures of Antigen Provide an Effective Means for Regulating Mast Cell Activation

Abstract

Nanostructures containing 2,4-Dinitrophenyl (DNP) as antigen were designed and produced to investigate antibody-mediated activation of mast cells. The design consists of nanogrids of DNP termini inlaid in alkanethiol self-assembled monolayers (SAMs). Using scanning probe-based nanografting, nanometer precision was attained for designed geometry, size and periodicity. Rat basophilic leukemia (RBL) cells exhibited high sensitivity to the geometry and local environment of DNP presented on these nanostructures. The impact included cellular adherence, spreading, membrane morphology, cytoskeleton structure, and activation. The highest level of spreading and activation was induced by nanogrids of 17 nm line width and 40 nm periodicity, with DNP haptens 1.4 nm above the surroundings. The high efficacy is attributed to two main factors. First, DNP sites in the nanostructure are highly accessible by anti-DNP-IgE during recognition. Second, the arrangement or geometry of DNP termini in nanostructures promotes clustering of FcεRI receptors that are pre-linked to IgE. The clustering effectively initiates Lyn-mediated signaling cascades, ultimately leading to the degranulation of RBL cells. This work demonstrates an important concept, that nanostructures of ligands provide new and effective cues for directing cellular signaling processes.

Engineered Nanostructures of Haptens Lead to Unexpected Formation of Membrane Nanotubes Connecting Rat Basophilic Leukemia Cells

“…16 This spatial arrangement brings Fc ε RI and Lyn into proximity. Lyn plays a positive role in this signaling pathway, 40 leading to phosphorylation of tyrosine residues within ITAMs in Fc ε RI.…”

“…17,18,42,43 Our prior work revealed that surface-bound antigens, when positioned at a matching geometry of closely packed Fc ε RI protein molecules, exhibit high potency in activating mast cells. 16 This evidence, collectively, suggests that the presentation of the antigen or hapten at the nanometer level dictates the arrangement of cellular receptors and thus impacts signaling pathways. Specifically, clustering (in 20 nm periodicity) of antigen such as DNP hepten is known to activate antibody-Fc ε RI-mediated activation among mast cells, while lack of or significant reduction of clustering hampers the activation signaling cascades.…”

“…Lyn plays a positive role in this signaling pathway, 40 leading to phosphorylation of tyrosine residues within ITAMs in Fc ε RI. 16,17,20 Phosphorylated ITAMs then serve as docking sites for Syk. 20,40 The binding of Syk results in activation of Syk and leads to downstream signaling pathways resulting in degranulation and F-actin reorganization.…”

“…45–53 The periodicity of this DNP grid nanostructure is 39±4 nm, and the edge-to-edge separation is 22 ± 5 nm. 16 (C) From the perspective of geometry only, arrangements of IgE-Fc ε RI complexes for discouraging degranulation and facilitating nanotube formation. (D) AFM topographic image of Nano2.…”

“…16 For example, arrays of nanogrids of antigen could activate mast cells to a higher degree than using soluble forms of antigen. 16 Using RBL cells, this work investigates if nanotechnology could facilitate cellular signaling cascades that are otherwise extremely difficult to activate, e.g ., formation of membrane nanotubes. Our results indicate that arrays of nanorings lead to the formation of membrane nanotubes among RBL cells.…”